title: Biomimetic assembly and activation of [FeFe]-hydrogenases.
abstract: Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux (CEA / Université Grenoble 1 / CNRS), 17 rue des Martyrs, F-38054 Grenoble cedex 9, France.|Collège de France, 11 place Marcelin-Berthelot, F-75231 Paris cedex 5, France.
authors.forename: G
authors.initials: G
authors.lastname: Berggren
authors.affiliation: Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Adamska
authors.affiliation: Lehrstuhl Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr Universität Bochum, Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Lambertz
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux (CEA / Université Grenoble 1 / CNRS), 17 rue des Martyrs, F-38054 Grenoble cedex 9, France.
authors.forename: T R
authors.initials: TR
authors.lastname: Simmons
authors.affiliation: Lehrstuhl Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr Universität Bochum, Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Esselborn
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux (CEA / Université Grenoble 1 / CNRS), 17 rue des Martyrs, F-38054 Grenoble cedex 9, France.
authors.forename: M
authors.initials: M
authors.lastname: Atta
authors.affiliation: CEA, INAC, LCIB (UMR-E 3 CEA / UJF-Grenoble 1), 17 rue des Martyrs, F-38054 Grenoble CEDEX 9, France.
authors.forename: S
authors.initials: S
authors.lastname: Gambarelli
authors.affiliation: CEA, INAC, LCIB (UMR-E 3 CEA / UJF-Grenoble 1), 17 rue des Martyrs, F-38054 Grenoble CEDEX 9, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Mouesca
authors.affiliation: Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Reijerse
authors.affiliation: Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Lubitz
authors.affiliation: Lehrstuhl Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr Universität Bochum, Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Happe
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux (CEA / Université Grenoble 1 / CNRS), 17 rue des Martyrs, F-38054 Grenoble cedex 9, France.
authors.forename: V
authors.initials: V
authors.lastname: Artero
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux (CEA / Université Grenoble 1 / CNRS), 17 rue des Martyrs, F-38054 Grenoble cedex 9, France.|Collège de France, 11 place Marcelin-Berthelot, F-75231 Paris cedex 5, France.
authors.forename: M
authors.initials: M
authors.lastname: Fontecave
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D040761:Biomimetic Materials / Q000138:chemical synthesis* / Q000378:metabolism*; D032701:Biomimetics; D020134:Catalytic Domain; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D046969:Clostridium acetobutylicum / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006864:Hydrogenase / Q000378:metabolism*; D008024:Ligands; D017550:Spectroscopy, Fourier Transform Infrared; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D001059:Apoproteins; D008024:Ligands; D006864:Hydrogenase